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Processing of the amyloid precursor protein (APP) and amyloid beta (Aß) has been for decades in the center of Alzheimer's disease (AD) research. Beside many other variables, lipids, especially cholesterol and its derivatives, are discussed to contribute to AD pathogenesis. Several studies show that cholesterol affects APP metabolism. Also the converse mechanism, the direct influence of Aß on cholesterol metabolism, has been described. To further investigate this crosstalk between cholesterol- and APP metabolism, a high-fat feeding study was conducted with animals overexpressing human APPSL and/or human ApoB-100. The impact of diet and genotype on cerebral cholesterol metabolism and content as well as spatial learning and memory was examined. While behavioral performance was not influenced by this high fat diet (HFD), reduction of cortical free cholesterol levels and mRNA expression patterns under normal diet and HFD conditions in human APPSL overexpressing mice argue for an important role of APP in cerebral lipid metabolism. From our results we conclude that increased APP metabolism in ApoBxAPP and APPSL mice induces mechanisms to reduce free cholesterol levels.
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Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Córtex Cerebral/metabolismo , Colesterol/metabolismo , Doença de Alzheimer/sangue , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Colesterol/sangue , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/metabolismo , Feminino , Genótipo , Homeostase , Humanos , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: ß-Synuclein (ß-Syn) is a member of the highly homologous synuclein protein family. The most prominent family member, α-synuclein (α-Syn), abnormally accumulates in so-called Lewy bodies, one of the major pathological hallmarks of α-synucleinopathies. Notably, parts of the peptide backbone, called the nonamyloid component, are also found in amyloid plaques. However, ß-Syn seems to have beneficial effects by reducing α-Syn aggregation, and amyloid antiaggregatory activity has been described. OBJECTIVE: The aim of the study was to analyze if wild-type ß-Syn can counteract functional and pathological changes in a murine Alzheimer model over different time periods. METHODS: At the onset of pathology, lentiviral particles expressing human ß-Syn were injected into the hippocampus of transgenic mice overexpressing human amyloid precursor protein with Swedish and London mutations (APPSL). An empty vector served as the control. Behavioral analyses were performed 1, 3 and 6 months after injection followed by biochemical and histological examinations of brain samples. RESULTS: ß-Syn expression was locally concentrated and rather modest, but nevertheless changed its effect on APP expression and plaque load in a time- and concentration-dependent manner. Interestingly, the phosphorylation of glycogen synthase kinase 3 beta was enhanced in APPSL mice expressing human ß-Syn, but an inverse trend was observed in wild-type animals. CONCLUSION: The initially reported beneficial effects of ß-Syn could be partially reproduced, but locally elevated levels of ß-Syn might also cause neurodegeneration. To enlighten the controversial pathological mechanism of ß-Syn, further examinations considering the relationship between concentration and exposure time of ß-Syn are needed.
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Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/metabolismo , beta-Sinucleína/metabolismo , Animais , Ansiedade/prevenção & controle , Linhagem Celular Tumoral , Modelos Animais de Doenças , Encefalite/metabolismo , Vetores Genéticos , Humanos , Lentivirus , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , alfa-Sinucleína/metabolismo , beta-Sinucleína/administração & dosagem , beta-Sinucleína/genéticaRESUMO
BACKGROUND: Beyond cognitive decline, Alzheimer's disease (AD) is characterized by numerous neuropathological changes in the brain. Although animal models generally do not fully reflect the broad spectrum of disease-specific alterations, the APPSL mouse model is well known to display early plaque formation and to exhibit spatial learning and memory deficits. However, important neuropathological features, such as neuroinflammation and lipid peroxidation, and their progression over age, have not yet been described in this AD mouse model. METHODS: Hippocampal and neocortical tissues of APPSL mice at different ages were evaluated. One hemisphere from each mouse was examined for micro- and astrogliosis as well as concomitant plaque load. The other hemisphere was evaluated for lipid peroxidation (quantified by a thiobarbituric acid reactive substances (TBARS) assay), changes in Aß abundance (Aß38, Aß40 and Aß42 analyses), as well as determination of aggregated Aß content (Amorfix A4 assay). Finally, correlation analyses were performed to illustrate the time-dependent correlation between neuroinflammation and Aß load (soluble, insoluble, fibrils), or lipid peroxidation, respectively. RESULTS: As is consistent with previous findings, neuroinflammation starts early and shows strong progression over age in the APPSL mouse model. An analyses of concomitant Aß load and plaque deposition revealed a similar progression, and high correlations between neuroinflammation markers and soluble or insoluble Aß or fibrillar amyloid plaque loads were observed. Lipid peroxidation, as measured by TBARS levels, correlates well with neuroinflammation in the neocortex but not the hippocampus. The hippocampal lipid peroxidation correlated strongly with the increase of LOC positive fiber load, whereas neocortical TBARS levels were unrelated to amyloidosis. CONCLUSIONS: These data illustrate for the first time the progression of major AD related neuropathological features other than plaque load in the APPSL mouse model. Specifically, we demonstrate that microgliosis and astrocytosis are prominent aspects of this AD mouse model. The strong correlation of neuroinflammation with amyloid burden and lipid peroxidation underlines the importance of these pathological factors for the development of AD. The new finding of a different relation of lipid peroxidation in the hippocampus and neocortical regions show that the model might contribute to the understanding of complex pathological mechanisms and their interplay in AD.
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Doença de Alzheimer , Precursor de Proteína beta-Amiloide/genética , Encefalite/etiologia , Hipocampo/patologia , Neocórtex/patologia , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite/patologia , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Vertebrate genes often play functionally distinct roles in different subsets of cells; however, tools to study the cell-specific function of gene products are poorly developed. Therefore, we have established a novel mouse model that enables the visualization and manipulation of defined subpopulations of neurons. To demonstrate the power of our system, we dissected genetic cascades in which Pax6 is central to control tangentially migrating neurons of the mouse brainstem. Several Pax6 downstream genes were identified and their function was analyzed by over-expression and knock-down experiments. One of these, Pou4f2, induces a prolonged midline arrest of growth cones to influence the proportion of ipsilaterally versus contralaterally settling neurons. These results demonstrate that our approach serves as a versatile tool to study the function of genes involved in cell migration, axonal pathfinding, and patterning processes. Our model will also serve as a general tool to specifically over-express any gene in a defined subpopulation of neurons and should easily be adapted to a wide range of applications.
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Neurônios/citologia , Rombencéfalo/citologia , Rombencéfalo/embriologia , Vertebrados/embriologia , Vertebrados/genética , Animais , Axônios/metabolismo , Movimento Celular/genética , Células Cultivadas , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Fenótipo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/genética , Fator de Transcrição Brn-3B/genética , Fator de Transcrição Brn-3B/metabolismoRESUMO
OBJECTIVES: Aggregation and misfolding of amyloid beta (Aß) and tau proteins, suggested to arise from post-translational modification processes, are thought to be the main cause of Alzheimer's disease (AD). Additionally, a plethora of evidence exists that links metabolic dysfunctions such as obesity, type 2 diabetes (T2D), and dyslipidemia to the pathogenesis of AD. We thus investigated the combinatory effect of T2D and human glutaminyl cyclase activity (pyroglutamylation), on the pathology of AD and whether astaxanthin (ASX) treatment ameliorates accompanying pathophysiological manifestations. METHODS: Male transgenic AD mice, APPxhQC, expressing human APP751 with the Swedish and the London mutation and human glutaminyl cyclase (hQC) enzyme and their non-transgenic (NTG) littermates were used. Both APPxhQC and NTG mice were allocated to 3 groups, control, T2D-control, and T2D-ASX. Mice were fed control or high fat diet ± ASX for 13 weeks starting at an age of 11-12 months. High fat diet fed mice were further treated with streptozocin for T2D induction. Effects of genotype, T2D induction, and ASX treatment were evaluated by analysing glycemic readouts, lipid concentration, Aß deposition, hippocampus-dependent cognitive function and nutrient sensing using immunosorbent assay, ELISA-based assays, western blotting, immunofluorescence staining, and behavioral testing via Morris water maze (MWM), respectively. RESULTS: APPxhQC mice presented a higher glucose sensitivity compared to NTG mice. T2D-induced brain dysfunction was more severe in NTG compared to the APPxhQC mice. T2D induction impaired memory functions while increasing hepatic LC3B, ABCA1, and p65 levels in NTG mice. T2D induction resulted in a progressive shift of Aß from the soluble to insoluble form in APPxhQC mice. ASX treatment reversed T2D-induced memory dysfunction in NTG mice and in parallel increased hepatic pAKT while decreasing p65 and increasing cerebral p-S6rp and p65 levels. ASX treatment reduced soluble Aß38 and Aß40 and insoluble Aß40 levels in T2D-induced APPxhQC mice. CONCLUSIONS: We demonstrate that T2D induction in APPxhQC mice poses additional risk for AD pathology as seen by increased Aß deposition. Although ASX treatment reduced Aß expression in T2D-induced APPxhQC mice and rescued T2D-induced memory impairment in NTG mice, ASX treatment alone may not be effective in cases of T2D comorbidity and AD.
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Doença de Alzheimer , Diabetes Mellitus Tipo 2 , Camundongos Transgênicos , Xantofilas , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Camundongos , Xantofilas/farmacologia , Xantofilas/metabolismo , Masculino , Doença de Alzheimer/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Humanos , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Dieta Hiperlipídica/efeitos adversos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Progressive accumulation of α-synuclein (α-Syn) protein in different brain regions is a hallmark of synucleinopathic diseases, such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. α-Syn transgenic mouse models have been developed to investigate the effects of α-Syn accumulation on behavioral deficits and neuropathology. However, the onset and progression of pathology in α-Syn transgenic mice have not been fully characterized. For this purpose we investigated the time course of behavioral deficits and neuropathology in PDGF-ß human wild type α-Syn transgenic mice (D-Line) between 3 and 12 months of age. RESULTS: These mice showed progressive impairment of motor coordination of the limbs that resulted in significant differences compared to non-transgenic littermates at 9 and 12 months of age. Biochemical and immunohistological analyses revealed constantly increasing levels of human α-Syn in different brain areas. Human α-Syn was expressed particularly in somata and neurites of a subset of neocortical and limbic system neurons. Most of these neurons showed immunoreactivity for phosphorylated human α-Syn confined to nuclei and perinuclear cytoplasm. Analyses of the phenotype of α-Syn expressing cells revealed strong expression in dopaminergic olfactory bulb neurons, subsets of GABAergic interneurons and glutamatergic principal cells throughout the telencephalon. We also found human α-Syn expression in immature neurons of both the ventricular zone and the rostral migratory stream, but not in the dentate gyrus. CONCLUSION: The present study demonstrates that the PDGF-ß α-Syn transgenic mouse model presents with early and progressive accumulation of human α-Syn that is accompanied by motor deficits. This information is essential for the design of therapeutical studies of synucleinopathies.
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Regulação da Expressão Gênica/genética , Proteínas de Filamentos Intermediários/metabolismo , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/metabolismo , Fatores Etários , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Proteínas de Filamentos Intermediários/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Crescimento Derivado de Plaquetas/genética , Fatores de TempoRESUMO
Introduction: Autism spectrum disorder (ASD) is a persistent neurodevelopmental condition characterized by two core behavioral symptoms: impaired social communication and interaction, as well as stereotypic, repetitive behavior. No distinct cause of ASD is known so far; however, excitatory/inhibitory imbalance and a disturbed serotoninergic transmission have been identified as prominent candidates responsible for ASD etiology. Methods: The GABA B receptor agonist R-Baclofen and the selective agonist for the 5HT7 serotonin receptor LP-211 have been reported to correct social deficits and repetitive behaviors in mouse models of ASD. To evaluate the efficacy of these compounds in more details, we treated BTBR T+ Itpr3 tf /J and B6.129P2-Fmr1 tm1Cgr /J mice acutely with R-Baclofen or LP-211 and evaluated the behavior of animals in a series of tests. Results: BTBR mice showed motor deficits, elevated anxiety, and highly repetitive behavior of self-grooming. Fmr1-KO mice exhibited decreased anxiety and hyperactivity. Additionally, Fmr1-KO mice's ultrasonic vocalizations were impaired suggesting a reduced social interest and communication of this strain. Acute LP-211 administration did not affect the behavioral abnormalities observed in BTBR mice but improved repetitive behavior in Fmr1-KO mice and showed a trend to change anxiety of this strain. Acute R-Baclofen treatment improved repetitive behavior only in Fmr1-KO mice. Conclusion: Our results add value to the current available data on these mouse models and the respective compounds. Yet, additional studies are needed to further test R-Baclofen and LP-211 as potential treatments for ASD therapy.
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Defective degradation and clearance of amyloid-ß as well as inflammation per se are crucial players in the pathology of Alzheimer's disease (AD). A defective transport across the blood-brain barrier is causative for amyloid-ß (Aß) accumulation in the brain, provoking amyloid plaque formation. Using primary porcine brain capillary endothelial cells and murine organotypic hippocampal slice cultures as in vitro models of AD, we investigated the effects of the antioxidant astaxanthin (ASX) on Aß clearance and neuroinflammation. We report that ASX enhanced the clearance of misfolded proteins in primary porcine brain capillary endothelial cells by inducing autophagy and altered the Aß processing pathway. We observed a reduction in the expression levels of intracellular and secreted amyloid precursor protein/Aß accompanied by an increase in ABC transporters ABCA1, ABCG1 as well as low density lipoprotein receptor-related protein 1 mRNA levels. Furthermore, ASX treatment increased autophagic flux as evidenced by increased lipidation of LC3B-II as well as reduced protein expression of phosphorylated S6 ribosomal protein and mTOR. In LPS-stimulated brain slices, ASX exerted anti-inflammatory effects by reducing the secretion of inflammatory cytokines while shifting microglia polarization from M1 to M2 phenotype. Our data suggest ASX as potential therapeutic compound ameliorating AD-related blood brain barrier impairment and inflammation.
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Doença de Alzheimer , Camundongos , Animais , Suínos , Doença de Alzheimer/metabolismo , Barreira Hematoencefálica/metabolismo , Peptídeos beta-Amiloides/metabolismo , Células Endoteliais/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Autofagia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Camundongos Transgênicos , Modelos Animais de DoençasRESUMO
Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are two common types of α-synucleinopathies and represent a high unmet medical need. Despite diverging clinical manifestations, both neurodegenerative diseases share several facets of their complex pathophysiology. Apart from α-synuclein aggregation, an impairment of mitochondrial functions, defective protein clearance systems and excessive inflammatory responses are consistently observed in the brains of PD as well as DLB patients. Leukotrienes are lipid mediators of inflammatory signaling traditionally known for their role in asthma. However, recent research advances highlight a possible contribution of leukotrienes, along with their rate-limiting synthesis enzyme 5-lipoxygenase, in the pathogenesis of central nervous system disorders. This review provides an overview of in vitro as well as in vivo studies, in summary suggesting that dysregulated leukotriene signaling is involved in the pathological processes underlying PD and DLB. In addition, we discuss how the leukotriene signaling pathway could serve as a future drug target for the therapy of PD and DLB.
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Doença por Corpos de Lewy , Doença de Parkinson , Sinucleinopatias , Encéfalo/metabolismo , Humanos , Leucotrienos , Doença de Parkinson/patologia , Transdução de Sinais , alfa-Sinucleína/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) still depicts an incurable and devastating disease. Drug development efforts are mostly based on superoxide dismutase 1 gene (SOD1)-G93A mice that present a very strong and early phenotype, allowing only a short time window for intervention. An alternative mouse model is available, that is based on the same founder line but has a reduced SOD1-G93A copy number, resulting in a weaker and delayed phenotype. To be able to use these SOD1-G93A/low mice for drug testing, we performed a characterization of ALS-typical pathologies. All analyses were performed compared to non-transgenic (ntg) littermates of the same sex and age. In vivo analysis of SOD1-G93A/low mice was performed by weekly body weight measurements, analysis of the survival rate, and measurement of the muscle strength of 24-30 weeks old female and male SOD1-G93A/low mice. Immunofluorescent labeling of SOD1, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba1) protein was performed in the cervical, thoracic, and lumbar ventral horn of the spinal cord of 24-30 weeks old male and female SOD1-G93A/low mice. The musculus gastrocnemius of male SOD1-G93A/low mice was labeled with fluorophore-conjugated α-bungarotoxin and antibodies against phosphorylated neurofilaments. Fluorescent labeling was detected and quantified by macro-based image analysis. Although SOD1 protein levels were highly increased in both sexes and all age groups, levels strongly peaked in 30 weeks old male SOD1-G93A/low mice. Astrocytosis and activated microglia in the spinal cord ventral horn and phosphorylated neurofilaments in the motor unit of the musculus gastrocnemius progressively increased, while muscle strength progressively decreased in male SOD1-G93A/low mice. In female SOD1-G93A/low mice, only activated microglia increased progressively, while muscle strength was constantly reduced starting at 26 weeks. These differences result in a shorter survival time of male SOD1-G93A/low mice of about 3 weeks compared to female animals. The results suggest that male SOD1-G93A/low mice present a stronger pathology and are, therefore, better suitable to evaluate the efficacy of new drugs against ALS as most pathological features are developing progressively paralleled by a survival time that allows treatment to start before symptom onset.
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Huntington's disease is known to be a purely genetic disease based on an expansion of a CAG base triplet repeat in the coding region of the Huntingtin gene. Some years ago, researchers were able to introduce the extensive full-length gene sequence of the mutant huntingtin gene into a rodent model. The resulting BACHD rat is already well characterized for behavioral deficits. So far, all analyses in this preclinical rat model were performed in male hemizygous animals. As homozygosity of transgenic models often causes an amplification of the phenotype and female HD patients present a stronger phenotype compared to men, we established a homozygous breeding colony and tested 2 and 5 months old homozygous male and female BACHD rats in a behavioral test battery. The tests included the grip strength test, Rota Rod, elevated plus maze, passive avoidance, and Barnes maze test. Our results show strong deficits in young female homozygous BACHD rats including increased body weight, motor deficits, muscle weakness, reduced anxiety and hypoactivity, as well as learning and memory deficits. Analysis of male homozygous BACHD rats showed only weak disease symptoms, similar compared to male hemizygous BACHD rats of already published studies. Evaluation of the breeding success showed that homozygous BACHD have a reduced number of pups at the time of birth that even decreases until weaning. Our results suggest that the phenotype of homozygous male BACHD rats barely differs from already published results of hemizygous BACHD rats while female homozygous BACHD rats display strong and early alterations.
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BACKGROUND: Preclinical Alzheimer's disease (AD) research strongly depends on transgenic mouse models that display major symptoms of the disease. Although several AD mouse models have been developed representing relevant pathologies, only a fraction of available mouse models, like the Tg4-42 mouse model, display hippocampal atrophy caused by the death of neurons as the key feature of AD. The Tg4-42 mouse model is therefore very valuable for use in preclinical research. Furthermore, metabolic biomarkers which have the potential to detect biochemical changes, are crucial to gain deeper insights into the pathways, the underlying pathological mechanisms and disease progression. OBJECTIVE: We thus performed an in-depth characterization of Tg4-42 mice by using an integrated approach to analyze alterations of complex biological networks in this AD in vivo model. METHODS: Therefore, untargeted NMR-based metabolomic phenotyping was combined with behavioral tests and immunohistological and biochemical analyses. RESULTS: Our in vivo experiments demonstrate a loss of body weight increase in homozygous Tg4-42 mice over time as well as severe impaired learning behavior and memory deficits in the Morris water maze behavioral test. Furthermore, we found significantly altered metabolites in two different brain regions and metabolic changes of the glutamate/4-aminobutyrate-glutamine axis. Based on these results, downstream effects were analyzed showing increased Aß42 levels, increased neuroinflammation as indicated by increased astro- and microgliosis as well as neuronal degeneration and neuronal loss in homozygous Tg4-42 mice. CONCLUSION: Our study provides a comprehensive characterization of the Tg4-42 mouse model which could lead to a deeper understanding of pathological features of AD. Additionally this study reveals changes in metabolic biomarker which set the base for future preclinical studies or drug development.
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Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Modelos Animais de Doenças , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , FenótipoRESUMO
BACKGROUND: To better understand the etiology and pathomechanisms of Alzheimer's disease, several transgenic animal models that overexpress human tau or human amyloid-beta (Aß) have been developed. In the present study, we generated a novel transgenic rat model by cross-breeding amyloid precursor protein (APP) rats with tau rats. We characterized this model by performing positron emission tomography scans combined with immunofluorescent labeling and cerebrospinal fluid analyses. METHODS: APP/Tau rats were generated by cross-breeding male McGill-R-Thy1-APP transgenic rats with female hTau-40/P301L transgenic rats. APP/Tau double transgenic rats and non-transgenic (ntg) littermates aged 7, 13, and 21 months were subjected to dynamic [11C] PiB scan and dynamic [18F]THK-5317 scans. For regional brain analysis, a template was generated from anatomical MR images of selected animals, which was co-registered with the PET images. Regional analysis was performed by application of the simplified reference tissue model ([11C]PiB data), whereas [18F]THK-5317 data were analyzed using a 2-tissue compartment model and Logan graphical analysis. In addition, immunofluorescent labeling (tau, amyloid) and cerebrospinal fluid analyses were performed. RESULTS: [11C]PiB binding potential (BPND) and [18F]THK-5317 volume of distribution (VT) showed an increase with age in several brain regions in the APP/Tau group but not in the ntg control group. Immunohistochemical analysis of brain slices of PET-scanned animals revealed a positive correlation between Aß labeling and [11C]PiB regional BPND. Tau staining yielded a trend towards higher levels in the cortex and hippocampus of APP/Tau rats compared with ntg littermates, but without reaching statistical significance. No correlation was found between tau immunofluorescence labeling results and the respective [18F]THK-5317 VT values. CONCLUSIONS: We thoroughly characterized a novel APP/Tau rat model using combined PET imaging and immunofluorescence analysis. We observed an age-related increase in [11C]PiB and [18F]THK-5317 binding in several brain regions in the APP/Tau group but not in the ntg group. Although we were able to reveal a positive correlation between amyloid labeling and [11C]PiB regional brain uptake, we observed relatively low human tau and amyloid fibril expression levels and a somewhat unstable brain pathology which questions the utility of this animal model for further studies.
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Doença de Alzheimer , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Animais , Feminino , Masculino , Tomografia por Emissão de Pósitrons , Ratos , Ratos TransgênicosRESUMO
Neurofilament-light chain (NF-L) is a well-known clinical biomarker of many neurodegenerative diseases. By analyzing amyotrophic lateral sclerosis (ALS) patients cerebrospinal fluid (CSF) or plasma, progression of NF-L levels can forecast conversion from the presymptomatic to symptomatic stage and time of survival. The use of plasma for NF-L measurement makes this biomarker exceptionally valuable for clinical studies since sample collection can be performed repeatedly without causing much harm. Detailed analyses of NF-L expression in neurodegenerative disease patient's samples were already performed, while NF-L levels of preclinical models of ALS, Alzheimer's and Parkinson's disease as well as lysosomal storage diseases are still widely unknown. We therefore evaluated NF-L levels in the plasma of the ALS models SOD1-G93A low expressor and TAR6/6 mice, the Alzheimer's disease (AD) model 5xFAD, the Parkinson's disease model Line 61 and the Gaucher disease (GD) model 4L/PS-NA and the CSF of selected models. Our results show that NF-L levels are highly increased in the plasma of ALS, Alzheimer's and GD models, while in the analyzed Parkinson's disease model NF-L plasma levels barely changed. Most analyzed models show a progressive increase of NF-L levels. NF-L measurements in the plasma of the neurodegenerative disease mouse models of ALS and AD are thus a good tool to evaluate disease progression. Compared to analyses in human tissues, our results suggest a high translation value of murine NF-L levels and their progression. Furthermore, our data indicate that NF-L might also be a good biomarker for disorders with a neuronal component like some lysosomal storage diseases.
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Excessive tau phosphorylation is the hallmark of tauopathies. Today's research thus focusses on the development of drugs targeting this pathological feature. To test new drugs in preclinical studies, animal models are needed that properly mimic this pathological hallmark. The htau mouse is a well-known model expressing human but lacking murine tau, allowing to evaluate the efficacy of tau modifying compounds without interference from murine tau. Htau mice are well-characterized for tau pathology at older age, although it is often not specified on which genetic background analyzed animals were bred. Since it was shown that the genetic background can influence the pathology, we evaluated the phosphorylation status of young and adult htau mice on a C57BL/6J background by analyzing ptau Ser202 and ptau Ser396 levels in the cortex and hippocampus of 3 and 12 month old animals by immunofluorescent labelling. Additionally, we evaluated total tau, ptau Thr231 and ptau Thr181 in the soluble and insoluble brain fraction of 3-15 month old htau mice by immunosorbent assay. Our results show that ptau levels of all analyzed residues and age groups are similar without strong increases over age. These data show that tau is already phosphorylated at the age of 3 months suggesting that phosphorylation starts even earlier. The early start of tau phosphorylation in htau mice enables the use of these mice for efficacy studies already at very young age.
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Huntington's disease (HD) is caused by an expansion of CAG triplets in the huntingtin gene, leading to severe neuropathological changes that result in a devasting and lethal phenotype. Neurodegeneration in HD begins in the striatum and spreads to other brain regions such as cortex and hippocampus, causing motor and cognitive dysfunctions. To understand the signaling pathways involved in HD, animal models that mimic the human pathology are used. The R6/2 mouse as model of HD was already shown to present major neuropathological changes in the caudate putamen and other brain regions, but recently established biomarkers in HD patients were yet not analyzed in these mice. We therefore performed an in-depth analysis of R6/2 mice to establish new and highly translational readouts focusing on Ctip2 as biological marker for motor system-related neurons and translocator protein (TSPO) as a promising readout for early neuroinflammation. Our results validate already shown pathologies like mutant huntingtin aggregates, ubiquitination, and brain atrophy, but also provide evidence for decreased tyrosine hydroxylase and Ctip2 levels as indicators of a disturbed motor system, while vesicular acetyl choline transporter levels as marker for the cholinergic system barely change. Additionally, increased astrocytosis and activated microglia were observed by GFAP, Iba1 and TSPO labeling, illustrating, that TSPO is a more sensitive marker for early neuroinflammation compared to GFAP and Iba1. Our results thus demonstrate a high sensitivity and translational value of Ctip2 and TSPO as new marker for the preclinical evaluation of new compounds in the R6/2 mouse model of HD.
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Huntington disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion in the HTT gene. Various HD animal models have been generated to mimic the motor, cognitive and neuropsychiatric disturbances that affect HD patients. Reproducing disease phenotypes within these models is essential to identify reliable readouts for therapy studies. We validated behavioral phenotypes shown earlier by other research groups in the BACHD rat model, using both previously applied and novel tests for motor, cognitive and anxiety-like behaviors. We first confirmed known BACHD rats' phenotypes in rotarod, open field (OF) and elevated plus maze (EPM) tests. We then assessed the reproducibility of key phenotypes in the model using new tests: cliff hanging, passive avoidance (PA), Morris water maze (MWM), light dark box and light spot tests. We confirmed impaired motor coordination in the rotarod test and reduced activity in the OF. In line with earlier results in BACHD rats using different tests, we showed impaired reversal learning in MWM and decreased anxiety-like behavior with the light spot test supporting the validity of BACHD rats as a model of HD. Results in the EPM, light dark box, cliff hanging and PA tests did not confirm earlier findings. This may depend on phenotype inconsistencies or rather be related to differences in environmental variables, test typology, experimental settings, animal age and chosen behavioral parameters.
Assuntos
Comportamento Animal , Modelos Animais de Doenças , Doença de Huntington/psicologia , Animais , Aprendizagem da Esquiva , Masculino , Aprendizagem em Labirinto , Fenótipo , Ratos , Ratos Sprague-Dawley , Teste de Desempenho do Rota-RodRESUMO
Senile plaques frequently contain Aß-pE(3), a N-terminally truncated Aß species that is more closely linked to AD compared to other Aß species. Tau protein is highly phosphorylated at several residues in AD, and specifically phosphorylation at Ser202/Thr205 is known to be increased in AD. Several studies suggest that formation of plaques and tau phosphorylation might be linked to each other. To evaluate if Aß-pE(3) and ptau Ser202/Thr205 levels correlate in human and transgenic AD mouse models, we analyzed human cortical and hippocampal brain tissue of different Braak stages as well as murine brain tissue of two transgenic mouse models for levels of Aß-pE(3) and ptau Ser202/Thr205 and correlated the data. Our results show that Aß-pE(3) formation is increased at early Braak stages while ptau Ser202/Thr205 mostly increases at later stages. Further analyses revealed strongest correlations between the two pathologies in the temporal, frontal, cingulate, and occipital cortex, however correlation in the hippocampus was weaker. Evaluation of murine transgenic brain tissue demonstrated a slow but steady increase of Aß-pE(3) from 6 to 12 months of age in the cortex and hippocampus of APPSL mice, and a very early and strong Aß-pE(3) increase in 5xFAD mice. ptau Ser202/Thr205 levels increased at the age of 9 months in APPSL mice and at 6 months in 5xFAD mice. Our results show that Aß-pE(3) and ptau Ser202/Thr205 levels strongly correlate in human as well as murine tissues, suggesting that tau phosphorylation might be amplified by Aß-pE(3).
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Proteínas tau/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fosforilação , Ácido Pirrolidonocarboxílico/química , Especificidade da Espécie , Proteínas tau/genéticaRESUMO
INTRODUCTION: Tau deposition is one of the hallmarks of Alzheimer's disease (AD) and can be visualized and quantified using [18F]THK-5317 together with kinetic modeling. To determine the feasibility of this approach, we measured blood/plasma pharmacokinetics and radiotracer metabolism in female and male rats. METHODS: Female and male rats (n = 11-12) were cannulated via the femoral artery for continuous blood sampling. Blood sampling was performed at regular intervals after intravenous injection of [18F]THK-5317. After collection of the last blood sample, animals were sacrificed, and organs were excised. Blood from minute 5, 20 and 60 was centrifuged to obtain plasma. Radiolabeled metabolites in plasma, brain, liver and urine were analyzed by radio-thin-layer chromatography (radio-TLC). RESULTS: Plasma pharmacokinetics and metabolism were significantly different between female and male rats. [18F]THK-5317 plasma clearance was faster in female (0.66 ± 0.08 mL/h/kg BW) than in male (0.52 ± 0.11 mL/h/kg BW) rats (p = .005). The percentage of unmetabolized parent was significantly different between both sexes at 20 min and 60 min p.i. In the liver, a 1.6-fold higher radioactivity concentration was found in male versus female animals and in addition also the percentage of unmetabolized parent was different. CONCLUSION: Our results show pronounced sex differences in blood/plasma pharmacokinetics and metabolism of [18F]THK-5317 in rats. Female animals showed a faster plasma clearance compared to males. These results underline the importance of investigating both sexes and also support the notion that individual input functions or sex-specific population-based input functions are needed for kinetic modeling analyses. ADVANCES IN KNOWLEDGE: First preclinical study in rats showing pronounced sex differences in blood/plasma pharmacokinetics and metabolism of [18F]THK-5317. IMPLICATIONS FOR PATIENT CARE: Sex-specific differences might also be present in humans and thus clinical trials should have adequate sample size to account for effects in men and women separately.
Assuntos
Compostos de Anilina/farmacocinética , Quinolinas/farmacocinética , Caracteres Sexuais , Doença de Alzheimer/metabolismo , Compostos de Anilina/sangue , Compostos de Anilina/metabolismo , Animais , Feminino , Masculino , Quinolinas/sangue , Quinolinas/metabolismo , Traçadores Radioativos , Ratos , Distribuição Tecidual , Proteínas tau/metabolismoRESUMO
Alzheimer's disease can be modelled by different transgenic mouse strains. To gain deeper insight into disease model mechanisms, the previously described Tg4-42 mouse was analysed for transgene integration. On RNA/DNA level the transgene integration resulted in more than 20 copy numbers and further caused a deletion of exon 2 of the retinoic acid receptor beta. These findings were also confirmed on protein level with highly decreased retinoic acid receptor beta protein levels in homozygous Tg4-42 mice and may have an impact on the previously described phenotype of homozygous Tg4-42 mice to be solely dependent on amyloid-ß 4-42 expression. Since hemizygous mice show no changes in RARB protein levels it can be concluded that the previously described phenotype of these mice should not be affected by the retinoic acid receptor beta gene knockout. In order to fully understand the results of transgenesis, it is extremely advisable to determine the genome integration site and the basic structure of the inserted transgenes. This can be carried out for instance by next-generation sequencing techniques. Our results thus suggest that a detailed characterization of new disease models using the latest genomics technologies prior to functional studies could be a valuable tool to avoid an unexpected genetic influence on the animals' phenotype that is not only based on the inserted transgene. This would also significantly improve the selection of mouse models that are best suited for therapeutic development and basic research.